PROJECT SUMMARY/ABSTRACT River blindness (onchocerciasis) and lymphatic filariasis are two major neglected diseases caused by parasitic nematodes that together affect an estimated 145 million people worldwide in mostly poor, developing countries. To date, there are no vaccines for these diseases and there are no drugs that are effective against the adult stages (macrofilariae) and amenable for mass drug distribution to eliminate these diseases. The overall goal of our project is to identify macrofilaricidal lead candidates for the treatment of river blindness and lymphatic filariasis. There is a critical need for safe, effective macrofilaricidal drugs for use in mass drug administration and test and treat settings to support international control programs working towards the goal of eliminating these diseases. Recently, we identified a potent compound that is effective in killing both adult Onchocerca and Brugia worms in the nanomolar range in vitro. We hypothesize that THP will be an effective macrofilaricide in our newly developed animal models of infection and that THP will significantly decrease worm burden in both of these infections. We have already synthesized analogs that might be even more potent in our in vitro screening assays and consequently in vivo. We further propose to identify THP target(s) in adult Onchocerca and Brugia using an innovative chemical-proteomic approach referred to as thermal shift (TS) profiling. By identifying the target(s) of our chemical probes, we may elucidate novel biological pathway(s) that are essential and relevant to other nematode parasites and pathogenic organisms. In the R33 phase, we will optimize the potency of THP and its analogs utilizing iterative cycles of chemical design and synthesis along with in vitro and in vivo evaluation. We will also use the thermal shift assay to confirm that these newly synthesized compounds are acting at the same target(s) identified in the R21 phase. Predicted targets identified from the TS assays with Brugia and Onchocerca worm lysates will be evaluated by RNAi to assess their essential functions during worm development and for survival. By the end of the R33, we will have identified 1-2 lead candidates and evaluated the drug targets in functional worm assays. Future directions will include further refinement of the 1-2 preclinical candidates in late phase lead optimization and formulation studies, followed by animal models of lymphatic filariasis (Brugia/gerbil infection) and the cow model of onchocerciasis to validate potency and efficacy as macrofilaricides.